Various analytical procedures and devices are commonly employed in assays to determine the presence and/or concentration of substances of clinical significance which may be present in biological fluids such as urine, whole blood, plasma, serum, sweat or saliva. Such substances are commonly referred to as analytes and can include specific binding partners, e.g. antibodies or antigens, drugs and hormones. One sort of test device is the so-called dipstick, containing enzymes or reagents which are interactive with the analyte and will interact with it in a manner which results in an indicator changing color which can be correlated with the presence or, in a semi-quantitative methods, the concentration of the analyte in the fluid sample. More recently there have been developed test strips which operate on the principle of immunochromatography in which labeled antibodies, specific for the analyte are applied to a strip of absorbant material through which the test fluid and labeled antibodies can flow by capillarity. By immobilizing analyte (or an analog thereof) in a particular portion of the strip, i.e. capture zone, and measuring the amount of labeled antibody which is captured through specific binding, the concentration of analyte in the test sample can be semi-quantitatively determined. This sort of assay, in which the label is an enzyme and there is placed a substrate for the enzyme in the capture zone to provide a colored response, is more fully described in U.S. Pat. No. 4,446,232. In U.S. Pat. No. 4,703,017 there is described a similar assay in which the label is a particulate material which, upon aggregation in the capture zone due to specific binding between the immobilized analyte and particle labeled antibody, provides a visible detectable response.
The clinical usefulness of analyses for various analytes can be enhanced by determining the concentration of a second analyte whose concentration in the biological fluid is clinically related to that of the first analyte. This is particularly true in urinalysis where the concentration of urine is very dependent on the degree of hydration, i.e. how hydrated the patient is as determined by fluid intake. In the case where a patient is dehydrated the urine is very concentrated and measurements tend to over predict an analyte's concentration which leads to false positive results. In the case of an overly hydrated patient, where the concentration of analytes is dilute, measurements tend to under predict the analyte's concentration leading to false negative results. Several analytes are known to measure urine concentration and are relatively unaffected by disease. The most notable example of the second analyte is creatinine, the end metabolite when creatine becomes creatine phosphate which is used as an energy source for muscle contraction and is, therefore, relatively constant for a given muscle mass. The creatinine produced is filtered by the kidney glomeruli and then excreted into the urine without reabsorption.
In order to increase the accuracy of urinary assays and minimize the problem of variable urine flow rates which result in urine dilution or concentration, analyte/creatinine ratios are used in urine protein assays to normalize the urine concentration. Common creatinine assays include the alkaline Jaffe and Benedict-Behre methods which are run at a high pH, typically in the range of from 11.5 to 12.5. More recently, there has been developed a creatinine assay in which the urine sample is contacted with cupric ions in the presence of citrate, a hydroperoxide and an oxidizable dye which provides a colored response in the presence of oxygen free radicals and a pseudoperoxide. Creatinine quantitation may also be accomplished immunologically as described in WO 96/34271. Those second analytes whose concentration in the sample of body fluid is clinically related to the concentration of the first analyte are not limited to creatinine in urine nor is urine the only body fluid which can be assayed by the method of the present invention. Thus, for example, the body fluid tested can be whole blood and the first analyte can be HbA.sub.1c with the second analyte being total hemoglobin since the apparent concentration of HbA.sub.1c can be adjusted to the whole blood's total hemoglobin concentration to factor out bias in the HbA.sub.1c assay. Inulin, administered intravenously, is, like creatinine, an indicator of renal flow. Typical of other first analytes which can be assayed in conjunction with creatinine as the second analyte are bacteria, red blood cells, leukocytes, various urinary proteins and glucose. The IgG concentration in urine can be corrected based on albumin as the second analyte.
In WO-96/34271 there is disclosed a device for determining a target (first) analyte and creatinine in a fluid test sample which device has an assay strip for the detection of creatinine and a second assay strip for the detection of the target analyte. The creatinine concentration may be determined colorimetrically or by the specific capture of labeled creatinine binding partners. The concentration of the target analyte is corrected based on the sample's concentration which correction can either be done manually or by means of a pre-programmed reflectance analyzer.
The prior art systems for determining the ratio of two analytes either involve a determination of the concentrations of both analytes followed by a determination of the ratio arithmetically or involve directly ratioing a response, such as color, due to the first analyte to the response due to the second analyte followed by converting the ratioed response to concentration values. This type of system is demonstrated in U.S. Pat. No. 5,385,847. The direct ratios of the colored responses is accomplished by first converting the colors into numerical values such as absorbance or reflectance for the first and second analytes. These numbers are divided and converted to a ratio result based on calibration. These systems for determining ratios are suitable in analytical procedures that have wide dynamic ranges due to small standard errors. The dynamic range is the useful range of analyte concentrations that the procedure is capable of measuring with accuracy. This imposes the requirement that the useful analytic range be determined is directly dependent on a method's accuracy. The bounds of the range are concentrations beyond which the method is not capable of measuring with accuracy. This is the concentration at which the procedure is not capable of determining an unknown concentration of analyte with reasonable certainty (typically &gt;80%) as being different from the boundary limit. A method with an accuracy of a certain concentration of analyte such as 30 mg/L albumin is not useful for determining albumin at that concentration because the probability of the method being correct would equal the probability of the method being wrong.
The dynamic range for a ratio determined by two analytical procedures is always less than the range of either of the procedures by themselves due to a reduction in accuracy. The ratio determination is less accurate due to the addition of errors from both individual methods causing the error for the ratio determination to be increased. The ratio accuracy is defined by variance (or the square of the standard deviation) where: EQU Vx/y=Vx=X V1/y+VxX.mu..sup.2 1/+V1/yX.mu..sup.2 x
in which V=variance; .mu.=expected value; x/y=ration x=first analyte and y=second analyte.
With quantitative procedures, the dynamic range is wider than concentrations which are expected to be measured. The dynamic range of the procedures measuring the first and second analytes are so wide that the loss of range upon determining the ratio does not impact the usefulness of the ratio result. A method with a dynamic range of 5 to 500 mg albumin/g creatinine is capable of measuring an albumin to creatinine concentration in the medically useful range of 10 to 300 mg/gm as shown in U.S. Pat. No. 5,305,847. With semi-quantitative procedures, the dynamic ranges are narrowed since the methods have less accuracy than those which are quantitative. Additional reduction of accuracy and dynamic range due to the ratio calculation can have a negative impact on the usefulness of the ratio result. A dynamic range of 30 to 300 mg albumin/g creatinine is not capable of providing medically useful information at the boundary limits of the range, i.e. 30 or 300 mg/g.